Novel polyphosphate:amp phosphotransferase

ABSTRACT

This invention relates to a novel polyphosphate: AMP phosphotransferase (PAP), a gene coding this PAP, and their use. The PAP has the following properties: (A) action: catalyzing of the following two reactions: 
 
NMP+PolyP (n) →NDP+PolyP (n-1)  
 
dNMP+POlyP (n) →dNDP+PolyP (n-1)  
(wherein NMP represents nucleoside monophosphate, NDP represents nucleoside diphosphate, dNMP represents deoxynucleoside monophosphate, dNDP represents deoxynucleoside diphosphate, n represents degree of polymerization of the polyphosphate which is an integer of up to 100); (B) substrate specificity: specific to AMP, GMP, IMP, dAMP, and dGMP, also acting with CMP, UMP, dCMP, and TMP; (C) molecular weight: about 55 to 56 Kd (kilodalton); and (D) specific activity: at least 70 units per 1 mg of enzyme protein,

TECHNICAL FIELD

This invention relates to a novel polyphosphate:AMP phosphotransferase,a gene coding for this phosphotransferase, and their use.

BACKGROUND ART

Recent progress in genetic engineering has enabled large scaleproduction of various enzymes at a low cost, and economical processusing an enzymatic reaction has also been enabled for the production ofphysiologically active substances of value that has traditionally beenproduced by means of bioconversion using a live bacterium, fermentation,or chemical synthesis.

In the meanwhile, an enzymatic reaction requiring energy as in the caseof phosphorylation and amination needs adenosine 5′-triphosphate (ATP)for its energy donor or phosphate donor. In the conventional microbialtransformation and fermentation, ATP has been supplied by themicroorganism employed, whereas, in the case of the enzymatic process,addition of ATP to the reaction system and development of efficient ATPregeneration system are required.

However, process of inexpensive ATP synthesis has not yet beenestablished and commercially available ATP is still very expensive. Inaddition, both the substrate and the enzyme used in the common ATPregeneration system, namely, the combination of phosphocreatine andphosphocreatine kinase, or acetyl phosphate and acetate kinase are veryexpensive, and their use has been unpractical and limited to thelaboratory level.

In contrast to such high price of the ATP, adenosine 5′-monophosphate(AMP) can be produced at a relatively low cost. ATP is currentlyproduced either by chemical synthesis or by using microorganism or yeastfrom AMP or adenine. Accordingly, development of an efficient ATPregeneration process has been highly awaited that can be used in theenzymatic reaction system using the ATP wherein the ATP is enzymaticallyproduced from the relatively inexpensive AMP and the consumed ATP isefficiently regenerated instead of adding the expensive ATP.

In constructing a practical ATP generation/regeneration system,selection of the phosphate donor used is also important, andpolyphosphate, which is inexpensive and stable, has been considered themost promising candidate of the phosphate donor. Enzymes which are knownto be involved in the metabolism of the polyphosphate and which also actwith adenosine nucleotide include polyphosphate kinase andpolyphosphate:AMP phosphotransferase (hereinafter abbreviated as “PAP”).

PAP is an enzyme which phosphorylates AMP to produce ADP by usingpolyphosphate as the phosphate donor (J. Bacteriol., 173,6484-6488(1991)). Zenhder et al. has reported that an ATPgeneration/regeneration system wherein AMP and polyphosphate are thesubstrates functions, when PAP obtained from Acinetobacter johnsonii ispartially purified, and the partially purified PAP is used incombination with adenylate kinase (Appl. Environ. Microbiol., 66,2045-2051(2000)). Kameda et al. has reported that, in the enzymaticreaction system wherein ATP is consumed to generate AMP, a systemwherein ATP is generated from AMP using polyphosphate as the phosphatedonor functions efficiently when the combination of the PAP fromMyxococcus xanthus and E. coli polyphosphate kinase is used.

However, PAP is present in the Acinetobacter johnsonii cell in anextremely small amount, and with regard to the use of the crude PAP suchas cell extract, a problem has been pointed out that contamination ofthe enzymes which decompose the substance involved in the reaction (AMP,ADP, ATP, reaction substrate and/or reaction product) may invite loss ofreaction efficiency. Such problem can be obviated by the use of highlypurified PAP instead of the crude enzyme. PAP, however, is veryunstable, and the purification procedure of PAP is far too complicatedto adopt such purified PAP into practical use.

In coping with such problem, the inventors of the present inventionestimated that such problem can be challenged by producing the PAP ofAcinetobacter johnsonii in a large amount using a recombinant DNAtechnique. However, the amino acid sequence of the enzyme and the genefor such enzyme have not been reported at all.

DISCLOSURE OF THE INVENTION

The inventors of the present invention have succeeded in themass-production of the PAP in E. coli by constructing a screening systemadapted for the gene cloning by PAP activity, and cloning the genecoding for the PAP by using the thus constructed screening system. Inthe analysis of the thus produced recombinant PAP, the inventors alsofound that the enzyme has an extremely high specific activity, andcombination of such enzyme with adenylate kinase enables construction ofan efficient ATP generation/regeneration system.

Unexpectedly, the inventors also found that the recombinant PAP has theactivity of phosphate-transfer using polyphosphate for the phosphatedonor to generate nucleoside diphosphate even if the nucleosidemonophosphate were not AMP or GMP, and this is a difference from theconventional PAP obtained from Acinetobacter johnsonii.

While nucleoside diphosphate has been generally found useful as astarting material for enzymatically synthesizing polynucleotide used inmedical or chemical commodities, its synthesis has been far from easy.In the case of bioconversion, the phosphorylation can not be terminatedat the diphosphate stage, and chemical phosphorylation has been the onlyusable way for the nucleoside diphosphate production. However, chemicalphosphorylation is associated with the problem of simultaneousoccurrence of the side reaction that results in the formation ofbyproducts, and isolation and purification of the target nucleosidediphosphate from the reaction solution has been extremely complicated.In view of such situation, development of an efficient process forsynthesizing the nucleoside diphosphate by enzymatic phosphorylation ofthe nucleoside monophosphate is highly awaited, and PAP has beenconceived as one candidate for the enzyme used in the enzymaticsynthesis.

However, Zehnder et al. has reported that, PAP from Acinetobacterjohnsonii is AMP-specific, and while some phosphate-transfer is foundfor GMP, no phosphotransfer at all was found for other nucleotides (CMP,UMP, and IMP) (Appl. Environ. Microbiol., 66, 2045-2051 (2000)), and ithas been conceived that, PAP can not be used in the synthesis ofnucleoside diphosphate by enzymatic phosphorylation of the nucleosidemonophosphate.

The inventors of the present invention have made an intensive studybased on the novel findings as described above, and completed thepresent invention. This invention relates to the PAP (the PAP of thepresent invention) having the physical and chemical properties asdescribed below:

(A) action: catalyzing the following two reactions:NMP+PolyP_((n))→NDP+PolyP_((n-1))dNMP+POlyP_((n))→dNDP+PolyP_((n-1))(wherein NMP represents nucleoside monophosphate, NDP representsnucleoside diphosphate, dNMP represents deoxynucleoside monophosphate,dNDP represents deoxynucleoside diphosphate, n represents degree ofpolymerization of the polyphosphate which is an integer of up to 100);

(B) substrate specificity: specific to AMP, GMP, IMP, dAMP, and dGMP,also acting with CMP, UMP, dCMP, and TMP;

(C) molecular weight: about 55 to 56 Kd (kilodalton); and

(D) specific activity: at least 70 units per 1 mg of enzyme protein.

This invention also relates to a PAP having the amino acid sequence ofSEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1 whereindeletion, substitution, or addition of one to several amino acids hasoccurred.

This invention also relates to a PAP gene encoding the amino acidsequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1wherein deletion, substitution, or addition of one to several aminoacids has occurred.

This invention also relates to a PAP gene having the nucleotide sequenceof SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 2 whereindeletion, substitution, or addition of one to several nucleotides hasoccurred.

This invention also relates to a DNA fragment which hybridizes with thegene as described above under stringent conditions, and which codes forthe polypeptide having PAP activity.

This invention also relates to a method for producing nucleosidediphosphate wherein the nucleoside diphosphate is enzymatically producedfrom nucleoside monophosphate by using the PAP of the present inventionas the enzyme and polyphosphate as a phosphate donor.

This invention also relates to a method for producing ATP wherein ATP isenzymatically produced from AMP by using two enzymes, namely, the PAP ofthe present invention and adenylate kinase as the enzyme andpolyphosphate as a phosphate donor.

This invention also relates to an ATP generation/regeneration systemcomprising AMP, polyphosphate, PAP, and adenylate kinase wherein the PAPused is the PAP of the present invention.

Finally, this invention also relates to a method for producing acompound by using an ATP-consuming enzymatic reaction, wherein ATP isregenerated from the AMP simultaneously with the enzymatic reaction byusing an ATP regeneration system comprising polyphosphate, PAP, andadenylate kinase, and wherein the PAP used is the PAP of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a restriction map of the DNA fragment of about 10 kb includingthe PAP gene obtained from Acinetobacter johnsonii strain 210A. The PAPgene is included in SacI-HpaI DNA fragment.

FIG. 2 shows the first half of the nucleotide sequence of the 2.5 kb DNAfragment including the PAP gene, and the first half of the amino acidsequence of the PAP.

FIG. 3 shows the second half of the nucleotide sequence of the 2.5 kbDNA fragment including the PAP gene, and the second half of the aminoacid sequence of the PAP.

FIG. 4 shows the results of the pH stability evaluation according to thePAP of the present invention.

FIG. 5 shows the results of the optimal pH evaluation according to thePAP of the present invention.

FIG. 6 shows the results of the thermal stability evaluation accordingto the PAP of the present invention.

FIG. 7 shows the results of the optimal temperature evaluation accordingto the PAP of the present invention.

FIG. 8 shows synthesis of ADP and ATP from AMP using polyphosphate forthe phosphate donor and using the PAP and adenylate kinase.

BEST MODE FOR CARRYING OUT THE INVENTION

(1) PAP of the Present Invention

The PAP of the present invention has the physical and chemicalproperties as described below. (Also, see the Examples as will bepresented later.)

(A) Action: PAP catalyzes the following two reactions:NMP+PolyP_((n))→NDP+PolyP_((n-1))dNMP+PolyP_((n))→dNDP+PolyP_((n-1))(wherein NMP represents nucleoside monophosphate, NDP representsnucleoside diphosphate, dNMP represents deoxynucleoside monophosphate,dNDP represents deoxynucleoside diphosphate, n represents degree ofpolymerization of the polyphosphate which is an integer of up to 100).

(B) Substrate specificity: PAP acts specifically to AMP, GMP, IMP, DAMP,and dGMP, and it also acts with CMP, UMP, dCMP, and TMP.

(C) Molecular weight: PAP has a molecular weight of about 55 to 56 Kd(kilodalton).

(D) Specific activity: PAP has a specific activity of at least 70 unitsper 1 mg of enzyme protein.

(E) Optimal pH: around pH 8.5.

(F) Optimal temperature: around 50° C.

(G) pH stability: around pH 7 to 9.

(H) Thermal stability: PAP is stable up to about 50° C.

The “unit” used herein is the unit measured under the followingconditions, and 1 unit corresponds to the activity of producing 1 μmoleof ADP at 37° C. in 1 minute.

<Measurement Conditions>

Sample enzyme is added to 50 mM Tris HCl buffer solution (pH 7.8)containing 20 mM magnesium chloride, 10 mM AMP, and polyphosphate (30 mMcalculated in terms of inorganic phosphate) and the reaction is allowedto proceed by incubating the temperature at 37° C., and the reaction isthereafter terminated by a heat treatment at 100° C. for 1 minute, andamount of ADP in the reaction solution is measured by high performanceliquid chromatography (HPLC).

The PAP of the present invention has the amino acid sequence representedby SEQ ID NO: 1, and in particular, the recombinant PAP produced byrecombinant DNA process is substantially pure in terms of enzymaticactivity, and such recombinant PAP is free from the AMP-degradingactivity which is disadvantageous for phosphorylation of the AMP.

The amino acid sequence may also be the one wherein deletion,substitution, modification, or addition of one to several amino acidshas occurred as long as the activity of catalyzing the reactions asdescribed above is retained. The deletion, substitution, modification,or addition of the amino acid sequence may be induced by site specificmutagenesis (for example, Proc. Natl. Acad. Sci. USA, 81,4662-5666(1984); Nucleic Acid Res. 10, 6487-6500(1982); Nature 316,601-605(1985)) or the like which were known in the art before the filingof the present invention. The PAP of the present invention also includesan enzyme which has a homology of at least 90%, and more preferably atleast 95% with the amino acid sequence of SEQ ID NO: 1 as long as theactivity of catalyzing the reactions as described above is retained.

The PAP of the present invention is prepared by cloning the gene codingfor the enzyme having the amino acid sequence shown in SEQ ID NO: 1,typically, the PAP gene having the nucleotide sequence shown in SEQ IDNO: 2 from Acinetobacter johnsonii, and using this gene. When explainedby referring to an exemplary case of the gene from Acinetobacterjohnsonii, FIGS. 2 and 3 show the sequencing result of the nucleotidesequence of the DNA fragment cleaved by SacI and HpaI in the restrictionmap of FIG. 1, and the sequence of nucleotide numbers 604 to 2031 inFIGS. 2 and 3 corresponds to the structural gene of PAP, and thissequence is the same as the nucleotide sequence shown in SEQ ID NO: 2.

In the present invention, a gene having the nucleotide sequence of SEQID NO: 2 wherein deletion, substitution, insertion, or addition of oneor plural nucleotides has occurred in such sequence; a gene whichhybridizes with such gene under stringent conditions; and a gene whichhas a homology of at least 90%, and more preferably at least 95% withthe nucleotide sequence shown in SEQ ID NO: 2 may also be employed aslong as the gene is capable of producing the PAP of the presentinvention.

As in the case of the amino acid sequence as described above, the genewherein deletion, substitution, insertion, or addition of single orplural nucleotides has occurred means the gene wherein nucleotides ofthe number that can be deleted, substituted, inserted, or added by thetechnique known in the art such as site specific mutagenesis has beendeleted, substituted, inserted, or added. The hybridization understringent conditions means the hybridization using a solution containing5×SSC (1×SSC corresponds to 8.76 g of sodium chloride and 4.41 g ofsodium citrate dissolved in 1 liter of water), 0.1% w/vN-lauroylsalcosine sodium salt, 0.02% w/v SDS, and 0.5% w/v blockingagent, under the reaction temperature conditions of about 60° C. forabout 20 hours.

In the present invention, use of a gene further comprising SD sequence(Shine-Dalgarno Sequence) in the upstream the gene coding for the PAP isalso favorable, since the yield of the enzyme can be markedly improvedby the use of such gene.

Cloning of such gene, preparation of the expression vector using thethus cloned DNA fragment, preparation of the PAP using such expressionvector, and the like are well known to those skilled in the field ofmolecular biology, and such process may be done, for example, by usingthe procedure described in “Molecular Cloning” (Maniatis et al. ed.,Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y. (1982)).

In an exemplary method, amino acid sequence on N-terminal, C-terminal,or the like of the PAP purified from a microorganism belonging to genusAcinetobacter may be partly sequenced by a known method, and theoligonucleotide corresponding to the thus determined sequence issynthesized. Then, the DNA fragment containing the gene coding for thePAP may be cloned from the chromosomal DNA of the bacterium belonging togenus Acinetobacter by using the thus synthesized oligonucleotide as theprobe. Alternatively, chromosomal DNA may be cleaved by usingappropriate restriction enzymes, and a genomic library may be producedby the method commonly used in the art. The resulting genomic librarymay be screened based on the PAP activity to thereby clone the targetgene.

There is, however, a fair risk that the PAP is inactivated when the PAPis purified to a high degree, and therefore, use of a screening systemusing the PAP activity is preferable. The PAP activity used in thescreening is preferably the activity of generating ATP from AMP by thecombination of the PAP with polyphosphate kinase (PPK). Morespecifically, PAP and PPK may be used for generation of the ATP usingAMP for the substrate and isotope-labeled polyphosphate for thephosphate donor to thereby detect the generation of the isotope-labeledATP.

The host used for the cloning is not particularly limited. Use of E.coli, however, is preferable in view of the working and handlingconvenience.

Expression system with high expression rate of the cloned gene may beconstructed by preparing a recombinant expression vector whereinexpression regulatory signals (transcription initiation signal andtranslation initiation signal) are ligated in the upstream of the gene,after identification of the coding region of the gene through the methodto sequence the nucleotide sequence of the cloned DNA fragment, forexample, Maxam-Gilbert (Methods in Enzymology, 65, 499(1980)), dideoxychain terminator method (Methods in Enzymology, 101, 20(1983)), or thelike.

In order to produce the PAP in a large amount in the heterologousmicroorganism, the expression regulatory signals used are desirabletranscription and translation initiation signals which can be regulatedby an artificial means, and which are strong signals enabling a dramaticincrease in the PAP yield. Examples of transcription initiation signalwhich can be used when the host is E. coli include lac promoter, trppromoter, and tac promoter (Proc. Natl. Acad. Sci. USA., 80, 21(1983),and Gene, 20, 231(1982)), and trc promoter (J. Biol. Chem., 260,3539(1985)).

Exemplary vectors which can be used include plasmid vector, phagevector, and other vectors, and use of a plasmid vector is desirablesince it can be amplified in the microorganism, it includes an adequatedrug-resistant marker and cleavage site for the predeterminedrestriction enzymes, and it is copied in a large number in themicroorganism. Examples of the plasmid vectors which can be used whenthe host is E. coli include pBR322 (Gene, 2, 95(1975)), pUC18, and pUC19(Gene, 33, 103(1985)).

The microorganism is transformed by using the thus produced recombinantvector. The microorganism used for the host is not particularly limitedas long as it has high safety and handling convenience, and E. coli,yeast, and other microorganisms commonly used in the DNA recombinanttechniques can be used as desired. Use of E. coli, however, isfavorable, and exemplary strains include K12, C600, JM105, JM109 (Gene,33, 103-119(1985)), and other strains normally used in the DNArecombinant experiments.

Various methods have been reported for use in the transformation of amicroorganism, and an adequate method may be selected depending on thetype of the microorganism used for the host. When E. coli is used forthe host, E. coli may be transformed by the treatment with calciumchloride followed by the introduction of the plasmid to the interior ofthe cell at low temperature (J. Mol. Biol., 53, 159 (1970)).

The resulting transformant is cultivated in a culture medium wherein themicroorganism is propagatable, and the cultivation is continued and theexpression of the cloned PAP gene is induced until a large amount of thePAP is accumulated in the microorganism. The transformant may becultivated by a method commonly used in the art using a culture mediumcontaining a carbon source, a nitrogen source, and other nutrientsources necessary for the propagation of the microorganism cultivated.For example, when E. coli is used for the host, the culture medium usedmay be those commonly used for the cultivation of E. coli such as 2xYTmedium (Methods in Enzymology, 100, 20 (1983)), LB medium, M9CA medium(Molecular Cloning, supra), and the cultivation may be conducted at anincubation temperature of 20 to 40° C. with aeration shaking. When aplasmid is used for the vector, an appropriate antibiotic agent(ampicillin, kanamycin, or the like corresponding to the drug-resistantmarker) of an appropriate amount is added to the culture medium tothereby prevent loss of the plasmid in the course of the cultivation.

When the expression of the PAP gene should be induced during thecultivation, the gene expression may be induced by the method commonlyused with the promoter used. For example, when the promoter used is lacpromoter or tac promoter, an expression inducing agent such asisopropyl-β-D-thiogalactopyranoside (hereinafter abbreviated to as IPTG)may be added at an appropriate amount in the intermediate period of thecultivation.

The cells are collected from the thus produced culture by membraneseparation, centrifugation, or the like. While the collected cells maybe used as PAP with no further treatment, the PAP used is preferably acell-free extract prepared by suspending the cells collected in anappropriate buffer solution, lyzing the cell by a physical treatmentusing ultrasonication, French press, or the like, or by an enzymatictreatment using lysozyme or the like, and removing the bacterial residueto obtain the extract. While cell-free extract may be used for theenzyme source with no further treatment since the cell-free extractcontains an abundant amount of PAP, the PAP used may also be a partiallypurified product or a purified product prepared by either one orcombination of two or more of heat treatment, ammonium sulfateprecipitation, dialysis, treatment using a solvent such as ethanol,various chromatographic treatments, and other treatments commonly usedin the enzyme purification.

(2) Use of PAP in the Present Invention

The thus prepared PAP of the present invention can be used in thesynthesis of nucleoside diphosphate or deoxynucleoside diphosphate, andin the synthesis or regeneration of the ATP.

First, the nucleoside 5′-monophosphate (NMP) or the deoxynucleoside5′-monophosphate (dNMP) used in the synthesis of nucleoside5′-diphosphate (NDP) or deoxynucleoside 5′-diphosphate (dNDP) may be acommercially available product, and it may be used at a concentration inthe range of, for example, 1 to 200 mM, and preferably 10 to 100 mM.

The polyphosphate used may also be a commercially available product, andit may be used at a concentration in the range of 1 to 1000 mM, andpreferably 10 to 200 mM calculated in terms of inorganic phosphate. Thedegree of polymerization (n) of the polyphosphate may be up to 100, andpreferably about 10 to 50.

The synthesis of the NDP or dNDP may be accomplished by the reactionwherein NMP or dNDP and polyphosphate are added to a buffer solution ata pH in the range of 4 to 9, and at least 0.001 unit/ml, and preferably0.001 to 10 units/ml of the PAP of the present invention is added to thesolution at 20° C. or higher, and preferably at 30 to 40° C. for about 1to 50 hours with optional stirring.

The thus produced NDP or dNDP may be isolated and purified by achromatographic process or other process known in the art.

The ATP synthesis may be accomplished by converting AMP to ADP, andthen, to ATP by using the PAP of the present invention and adenylatekinase in the presence of polyphosphate.

The AMP added to the reaction solution may be a commercially availableproduct, and it may be used at an adequate concentration selected fromthe range of, for example, 1 to 200 mM, and preferably 10 to 100 mM.

The polyphosphate added may also be a commercially available product,and it may be used at a concentration selected from the range of 1 to1000 mM, and preferably 10 to 200 mM calculated in terms of inorganicphosphate. The degree of polymerization (n) of the polyphosphate may beup to 100, and preferably about 10 to 50.

The synthesis of the ATP may be accomplished by adding AMP andpolyphosphate in an appropriate buffer solution at pH in the range of 4to 9, and further adding at least 0.001 unit/ml, and preferably 0.001 to10 units/ml of the PAP of the present invention and at least 0.01unit/ml, and preferably 0.01 to 100 units/ml or more of adenylate kinaseto allow the reaction to proceed at 20° C. or higher, and preferably at30 to 40° C. for about 1 to 50 hours with optional stirring.

The thus generated ATP may be isolated and purified by a chromatographicmethod or other method known in the art.

The unit of the adenylate kinase activity is the one measured andcalculated by the procedure as described below. Sample enzyme is addedthe 50 mM Tris HCl buffer solution (pH 7.8) containing 10 mM magnesiumchloride, 10 mM AMP, and 10 mM ATP, and the solution is incubated at 37°C. to promote the reaction, and the reaction is terminated by a 1 minuteheating at 100° C. Amount of the ADP in the reaction solution ismeasured by using HPLC, and the activity of producing 2 μmole of ADP at37° C. in 1 minute is designated 1 unit.

The ATP regeneration/regeneration system comprising the AMP, thepolyphosphate, the PAP of the present invention, and the adenylatekinase may be used in detecting a minute amount of ATP in the course ofdetecting invisible microorganisms to check the cleanliness of the foodin the food plant, or in the detection of adenine nucleotide bybioluminescence, which is applicable in the measurement of freshness ofmeat, fish, vegetable, and other foods (see, for example, WO01/53513).

The ATP regeneration system comprising polyphosphate, the PAP of thepresent invention, and adenylate kinase is also applicable to theproduction of a compound using an ATP-consuming enzymatic reaction forallowing the ATP regeneration from the AMP to take place simultaneouslywith the enzymatic synthesis of the target compound thereby improvingthe efficiency of the synthesis.

Exemplary non-limited enzymatic reactions which can be combined withsuch ATP regeneration system include galactose-1-phosphate synthesissystem using galactokinase, UDP synthesis system using UMP kinase, andphosphocholine synthesis system using choline kinase, and the ATPregeneration system may be applied to any ATP-consuming enzymaticreaction.

The reaction conditions of the ATP synthesis system and the enzymaticreaction may be adequately determined by conducting tests in smallerscale, and the isolation and the purification of the target compound maybe accomplished by a method known in the art.

EXAMPLES

Next, the present invention is described in further detail by referringto the Examples, which by no means limit the scope of the invention. Inthe Examples, preparation of the DNA, cleavage with the restrictionenzyme, DNA ligation by using T4 DNA ligase, and transformation with theDNA were all conducted in accordance with “Molecular cloning II”(Sambrook et al. ed., Cold spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1989)). The restriction enzyme, AmpliTaq DNA polymerase, T4 DNAligase, and other DNA related enzymes were all obtained from TakaraShuzo K. K. Quantitative measurement of the nucleotides were conductedby HPLC, and more specifically, ODS-AQ312 column manufactured by YMC wasused for separation purpose with the eluent of 0.5M monopotassiumphosphate solution.

Example 1 Preparation of the PAP of the Present Invention

(1) Cloning of the PAP gene of Acinetobacter johnsonii Strain 210

(1-1) E. coli Polyphosphate Kinase and Isotope-Labeled Polyphosphate

E. coli polyphosphate kinase was prepared by the method described in thedocument (J. Biol. Chem., 268, 633-639(1993)). Isotope-labeledpolyphosphate was prepared using the thus prepared E. coli polyphosphatekinase according to the method of Akiyama et al. (J. Biosci. Bioeng.,91, 557-563(2001)).

(1-2) Production of Genomic Library of Acinetobacter johnsonii and itsScreening

Acinetobacter johnsonii strain 210A was inoculated in LB medium, and thecultivated overnight at 30° C. with stirring. The cells were collectedby centrifugation, and chromosomal DNA was purified. The chromosomal DNAof Acinetobacter johnsonii was partially decomposed with restrictionenzyme Sau3AI, and the fragments were fractionated by sucrose densitygradient centrifugation to recover the fraction of about 7 to 10 Kb.This DNA fragment and the plasmid vector pBlueScript SK(+) (purchasedfrom Toyobo) which had been cleaved with BamHI were ligated by using T4DNA ligase, and E. coli strain JM109 (purchased from Takara Shuzo) wastransformed with the solution of this DNA. The thus produced 6000ampicillin-resistant transformants were divided into groups of 50transformants.

After cultivating each group overnight at 37° C. in LB medium, the cellswere collected by centrifugation and washed with 20 mM Tris-HCl (pH8.0), and again suspended in the same buffer solution. BugBuster(purchased from Takara Shuzo) was added to the cell suspension at theequal amount, and the suspension was allowed to stand at roomtemperature for 30 minutes for cell lysis. Three volumes of 20 mMTris-HCl (pH 8.0) was then added to prepare the cell extract.

To 20 μl of the activity detecting solution (50 mM Tris HCl (pH 8.0), 40mM (NH4)₂SO₄, 4 mM MgCl₂, and 1 mM AMP) containing the isotope-labeledpolyphosphate (0.24 mM calculated in terms of phosphate) prepared abovewas added 1 μl of the cell extract, and the reaction was allowed toproceed at 37° C. for 1 hour. The reaction solution was then subjectedto thin layer chromatography (using developer: 0.75M KH₂PO₄, pH 3.5),and the ADP formation was detected on phospho-image analyzer BASS2000(manufactured by Fujix) for screening of the transformants. PAP activitywas detected for 1 clone in the 6000 strains.

Plasmid pPAP2 having the PAP gene of Acinetobacter johnsonii strain 210Ainserted therein was obtained from the resulting clone (FIG. 1). It isto be noted that the plasmid pPAP2 had inserted therein the chromosomalDNA of Acinetobacter johnsonii strain 210A which is about 10 kb, and itwas internationally deposited on the basis of Budapest treaty on May 22,2002 with the designation of plasmid DNA (pPAP2) to The NationalInstitute of Advanced Industrial Science and Technology (an IndependentAdministrative Institution), International Patent Organism Depositary(Chuo-6, 1, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan (Post code,305-8566)) with the accession number of FERM BP-8047.

(1-3) Analysis of PAP Gene of Acinetobacter johnsonii Strain 210

The DNA of Acinetobacter johnsonii strain 210A of 10 kb inserted in thepPAP2 was subcloned in various plasmids to determine the PAP activity ofthese transformants as described above, and it was then found that thePAP gene is located in SacI-HpaI DNA fragment of about 2.5 kb (FIG. 1).The nucleotide sequence of this DNA fragment was determined by dideoxychain terminator method (Science, 214, 1295(1981)), and it was thenfound that the PAP gene codes for the polypeptide (molecular weight:55.8 kd) comprising 475 amino acids (FIGS. 2 and 3).

(2) Preparation of Acinetobacter johnsonii PAP

The E. coli JM109 carrying the plasmid pPAP2 was cultivated overnight at28° C. in 2xYT medium containing ampicillin at 100 μg/ml. The cells werecollected by centrifugation, and suspended in the buffer solutioncomprising 50 mM Tris HCl (pH 7.8) and 1 mM EDTA. After ultrasonication,cell extract was collected by centrifugation. In the measurement of theextract for the PAP activity, it was found that PAP was produced at arate of 18.1 unit per 1 ml of the culture solution, and that theactivity was about 9000 folds higher than that of the contrast (the E.coli JM109 carrying no the plasmid).

It is to be noted that this productivity corresponds to about 150 timesthe productivity of the Acinetobacter Johnsonii. PAP was partiallypurified by fractionating the extract by ion exchange chromatographyusing DEAE TOYOPEARL 650M (TOSO) (eluent: 50 mM Tris HCl (pH 7.8),concentration gradient of 0 to 0.5M NaCl), and the collected fractionwas used for the enzyme sample. The specific activity of the PAP in thecollected fraction was 80.5 units/mg protein.

(3) Analysis of Various Properties of PAP

(3-1) Synthesis of Various NDP (Analysis of Substrate Specificity)

To 50 mM Tris HCl buffer solution (pH 8.0) containing 100 mM MgCl₂,polyphosphate (10 mM calculated in terms of inorganic phosphate), and 5mM of various NMP or dNMP was added PAP at various concentration, andthe solution was incubate at 37° C. for 10 minutes. The reaction wasterminated by heat treatment at 100° C. for 1 minute, and the solutionafter the reaction was subjected to HPLC to measure the amount of theNDP or the dNDP produced. The specific activity of each NMP or dNMP isshown in Table 1 as a relative value in relation to the specificactivity of the PAP in the phosphorylation of the AMP, which is assumed100%. TABLE 1 Specific activity Substrate (relative value) AMP 100%  GMP 10   CMP 0.09 UMP 0.13 IMP 2.2  dAMP 18   dGMP 2.6  dCMP  0.008 TMP 0.012(3-2) pH Stability

PAP was incubated in either 50 mM maleate or 50mM Tris HCl buffersolution of various pH at 37° C. for 10 minutes in the presence of 100mM magnesium chloride to measure the residual activity. The residualactivity was measured by allowing the reaction to take place at 37° C.for 10 minutes in the presence of 50 mM Tris buffer solution (pH 8.0),100 mM magnesium chloride, 5 mM AMP, and polyphosphate (10 mM calculatedin terms of inorganic phosphate), and measuring the resulting ADP byHPLC.

As shown in FIG. 4, the enzyme of the present invention was found toexhibit an enzymatic activity of 80% or more at a pH in the range of 7to 9 when the enzymatic activity at pH 8 was 100%.

(3-3) Optimal pH

The reaction was allowed to proceed in either 50 mM maleate or 50 mMTris HCl buffer solution of various pH at 37° C. for 10 minutes in thepresence of 100 mM magnesium chloride, polyphosphate (10 mM calculatedin terms of inorganic phosphate), and 5 mM AMP to measure the amount ofthe ADP produced by HPLC.

As shown in FIG. 4, the optimal pH of the enzyme of the presentinvention was approximately 8.5.

(3-4) Thermal Stability

PAP was incubated for 10 minutes in 50 mM Tris HCl buffer solution (pH8.0) containing 100 mM magnesium chloride in the presence or absence ofpolyphosphate (10 mM calculated in terms of inorganic phosphate) in thebath at various temperatures to measure the residual activity by theprocedure as described above.

As shown in FIG. 6, the enzyme of the present invention was found to bestable up to approximately 50° C. in the presence of the polyphosphate.

(3-5) Optimal Temperature

PAP was incubated for 10 minutes in 50 mM Tris HCl buffer solution (pH8.0) containing 100 mM magnesium chloride in the presence ofpolyphosphate (10 mM calculated in terms of inorganic phosphate) and 5mM AMP in the bath at various temperatures, and the enzymatic activitywas evaluated by measuring the amount of ADP produced by HPLC.

As shown in FIG. 7, optimal reaction temperature of the enzyme of thepresent invention was found to be approximately 50° C.

Example 2 Synthesis of ATP Using PAP and Adenylate Kinase

(1) Preparation of E. coli Adenylate Kinase

E. coli adenylate kinase was prepared by the procedure described in thedocument (Proc. Natl. Acad. Sci. USA, 97, 14168-14171 (2000)). However,the cell extract produced by ultrasonication was used for the enzymesolution, and the specific activity of the adenylate kinase in theenzyme solution was 12.5 units/mg protein.

(2) Synthesis of ATP

To 50 mM Tris HCl buffer solution (pH 7.8) containing 20 mM MgCl₂,polyphosphate (30 mM calculated in terms of inorganic phosphate), and 10mM AMP were added 1.5 units/ml of PAP and 0.4 unit/ml of adenylatekinase, and the solution was incubated at 37° C. for 60 minutes. Afterthe completion of the reaction, amount of the nucleotide in the reactionsolution was measured by HPLC.

As shown in FIG. 8, it was confirmed that AMP is promptly phosphorylatedby the PAP to produce ADP, and the thus produced ADP is promptlyconverted to ATP and AMP by the adenylate kinase that is also present inthe system, the repetition of this cycle resulting in the ATPaccumulation.

Example 3 Synthesis of Galactose-1-Phosphate Using ATP RegenerationSystem Comprising the combination of PAP and Adenylate Kinase

(1) Preparation of E. coli Galactokinase

E. coli strain JM109 having the plasmid pDR540 containing E. coligalactokinase gene (Gene, 20, 231(1982), obtained from Pharmacia) wasinoculated in 2xYT medium containing 100 μg/ml ampicillin, and the E.coli was cultivated at 37° C. with shaking. When the cell densityreached 4×10⁸/ml, IPTG was added to the culture medium to a finalconcentration of 1 mM, and the incubation was continued at 30° C. foranother 5 hours. After completion of the cultivation, the cells werecollected by centrifugation and suspended in 30 ml buffer solution (50mM Tris HCl (pH 7.8), 1 mM EDTA). The cells were lyzed byultrasonication, and bacterial residue was removed by furthercentrifugation. The solution collected was subjected to ion exchangechromatography using DEAE Toyopearl 650M (Toso) (eluent: 50 mM Tris HCl(pH 7.8), concentration gradient of 0 to 0.5M NaCl) for fractionationand partial purification of the galactokinase. The thus recoveredfraction was used for the enzyme solution of galactokinase. The specificactivity of the galactokinase in the enzyme solution was 6.5 units/mgprotein.

The unit of the glactokinase activity was the one measured andcalculated by the procedure as described below. Sample enzyme was addedthe 100 mM Tris HCl buffer solution (pH 7.8) containing 5 mM MgCl₂, 10mM ATP, and 10 mM galactose, and the solution was incubated at 37° C. topromote the reaction, and the reaction was terminated by a 1 minute heattreatment at 100° C. Amount of the galactose-1-phosphate in the reactionsolution was measured by using a sugar analyzer (Dionex), and theactivity of producing 1 μmole of galactose-1-phosphate at 37° C. in 1minute was designated 1 unit.

(2) Synthesis of Galactose-1-Phosphate

To 50 mM Tris HCl buffer solution (pH 7.8) containing 20 mM MgCl₂,polyphosphate (30 mM calculated in terms of inorganic phosphate), 5 mMAMP, and 50 mM (d) galactose were added 0.2 unit/ml PAP and 0.2 unit/mladenylate kinase, and then, galactokinase to 0.5 unit/ml, and thesolution was incubated at 37° C. for 8 hours. During the reaction,polyphosphate was also added respectively at 2 hours and 4 hours afterthe start of the reaction to 20 nM calculated in terms of inorganicphosphate. The reaction solution was analyzed with a sugar analyzer(Dionex), and production of 37.8 mM galactose-1-phosphate was confirmed.

Example 4 Synthesis of Nucleotide Diphosphate

(1) Synthesis of Various NDP

To 50 mM Tris HCl buffer solution containing 100 mM MgCl₂, polyphosphate(30 mM calculated in terms of inorganic phosphate), and 10 mM of an NMP(AMP, GMP, CMP, UMP, or IMP) was added PAP at 16 units/ml, and thesolution was incubated at 37° C. for 30 minutes. The results of the HPLCanalysis of the reaction solution are shown in Table 2. It is to benoted that no NDP generation was found when cell extract of E. coliJM109 was used for the contrast. TABLE 2 Substrate NDP generated AMP6.76 mM ADP GMP 7.16 mM GDP CMP 0.98 mM CDP UMP 0.85 mM UDP IMP 6.75 mMIDP(2) Enzymatic Synthesis of IDP

To 50 mM Tris HCl buffer solution containing 100 mM MgCl₂, polyphosphate(65 mM calculated in terms of inorganic phosphate), and 40 mM IMP wasadded PAP at 16 units/ml, and the solution was incubated at 37° C. for19 hours. The reaction solution was analyzed by HPLC, and formation of21. 2 mM IDP was confirmed.

INDUSTRIAL APPLICABILITY

This invention provides a novel PAP and its gene. This invention hasalso enabled a large scale production of the PAP with no difficulty,which could not have been done by the conventional technology. Anefficient synthesis and regeneration of ATP from AMP at a low cost hasbeen realized, and combination of this with an ATP-consuming enzymaticreaction system enables regeneration of the ATP consumed in the reactionsystem to facilitate efficient synthesis of the target compound.

In contrast to the conventional PAP, the PAP of the present inventionexhibits phosphorylation activity for various types of nucleoside5′-monophosphate other than AMP and deoxynucleoside 5′-monophosphate,and this enables smooth production of various types of nucleoside5′-diphosphate and deoxynucleoside 5′-diphosphate by an enzymaticprocess.

1. A polyphosphate:AMP phosphotransferase having the physical andchemical properties as described below: (A) action: catalyzing of thefollowing two reactions:NMP+PolyP_((n))→NDP+PolyP_((n-1))dNMP+PolyP_((n))→dNDP+PolyP_((n-1)) (wherein NMP represents nucleosidemonophosphate, NDP represents nucleoside diphosphate, dNMP representsdeoxynucleoside monophosphate, dNDP represents deoxynucleosidediphosphate, n represents degree of polymerization of the polyphosphatewhich is an integer of up to 100); (B) substrate specificity: specificto AMP, GMP, IMP, DAMP, and dGMP, also acting with CMP, UMP, dCMP, andTMP; (C) molecular weight: about 55 to 56 Kd (kilodalton); (D) specificactivity: at least 70 units per 1 mg of enzyme protein, with the provisothat 1 unit corresponds to the activity capable of forming 1 μmole ofADP in 1 minute at 37° C. measured under the following conditions:<measurement conditions> enzyme sample is added to 50 mM Tris HCl buffersolution (pH 7.8) containing 20 mM magnesium chloride, 10 mM AMP, andpolyphosphate (30 mM calculated in terms of inorganic phosphate) and thereaction is allowed to proceed by maintaining the temperature at 37° C.,and the reaction is thereafter terminated by a heat treatment at 100° C.for 1 minute, and amount of ADP in the reaction solution is measured byhigh performance liquid chromatography (HPLC).
 2. A polyphosphate:AMPphosphotransferase having the amino acid sequence of SEQ ID NO: 1 or theamino acid sequence of SEQ ID NO: 1 wherein deletion, substitution, oraddition of one to several amino acids has occurred.
 3. Apolyphosphate:AMP phosphotransferase gene encoding the amino acidsequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 1wherein deletion, substitution, or addition of one to several aminoacids has occurred.
 4. A gene according to claim 3 wherein thepolyphosphate: AMP phosphotransferase gene has the nucleotide sequenceof SEQ ID NO: 2 or the nucleotide sequence of SEQ ID NO: 2 whereindeletion, substitution, or addition of one to several nucleotides hasoccurred.
 5. A DNA fragment which hybridizes with the gene of claim 3 or4 under stringent conditions, and which codes for the polypeptide havingpolyphosphate:AMP phosphotransferase activity.
 6. A method for producingnucleoside diphosphate wherein the nucleoside diphosphate isenzymatically produced from nucleoside monophosphate by usingpolyphosphate:AMP phosphotransferase of claim 1 or 2 for the enzyme andpolyphosphate as a phosphate donor.
 7. A method for producing ATPwherein ATP is enzymatically produced from AMP by using two enzymes,namely, the polyphosphate:AMP phosphotransferase of claim 1 or 2 andadenylate kinase for the enzyme and polyphosphate as a phosphate donor.8. An ATP generation/regeneration system comprising AMP, polyphosphate,polyphosphate:AMP phosphotransferase, and adenylate kinase wherein thepolyphosphate:AMP phosphotransferase used is the enzyme of claim 1 or 2.9. A method for producing a compound by using an ATP-consuming enzymaticreaction, wherein ATP is regenerated from the AMP simultaneously withthe enzymatic reaction by using an ATP regeneration system comprisingpolyphosphate, polyphosphate:AMP phosphotransferase, and adenylatekinase, and wherein the polyphosphate:AMP phosphotransferase used is theenzyme of claim 1 or 2.